Technical Field of the Invention

The present invention is directed to oil-in-water (o/w) emulsions. In particular, the present invention is directed to oil-in-water emulsions that are resistant to oxidisation.

Background to the Invention

A wide variety of consumer goods contain oil-in-water emulsions including cosmetic preparations (eg skin creams, moisturisers, lotions, and hair and skin conditioning agents) and food products (eg dressings, ice creams, mayonnaises, spreads and sauces). The physio-chemical properties of the emulsions are critical for ensuring consumer acceptance of these products and furthermore the stability of the emulsion and of the ingredients therein is vital for ensuring the shelf-life of such products.

There are a number of mechanisms that degrade the quality of a product comprising an oil-in-water emulsion. Flocculation is the process by which particles in the emulsion are caused to clump together which may then float to the top of the continuous phase or settle to the bottom of the continuous phase. Creaming is the migration of a substance in an emulsion, under the influence of buoyancy, to the top of a sample while the particles of the substance remain separated. Breaking and coalescence is where the particles coalesce and form a layer within the continuous phase. Unstable emulsions are particularly susceptible to these mechanisms and suffer a break down in the physio-chemical structure of the emulsion and the loss of the beneficial properties required by consumers. The quality of a product comprising an oil-in-water emulsion can be further affected through the degradation of the oil. Oxidation is one such process that may cause deg radation and ca n lead to ra ncid ity and the loss of important functional ingredients. In their paper, Askolin et al. (Biomacromolecules, 2006, 7 (4), 1295-1301) disclose that olive oil and paraffin were emulsified in an aqueous hydrophobin solution by sonication however, this paper does not deal with the prevention of oxidation and moreover the emulsions were not stable.

There therefore remains a need for oil-in-water emulsions with improved shelf-lives that are resistant to oxidisation of oil therein. Tests and Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg colloid chemistry).

ON

As used herein the term "oil" is used as a generic term for lipids, fats or any mixture thereof, either pure or containing compounds in solution. Oils can also contain particles in suspension.

Lipids

As used herein the term "lipids" is used as a generic term for long chain fatty acids or long chain alcohols wherein the term "long chain" is used as a generic term for 12 carbon atoms or more.

Fats

As used herein the term "fats" is used as a generic term for compounds containing more than 80% triglycerides. They can also contain diglycerides, monoglycerides and free fatty acids. In common language, liquid fats are often referred to as oils but herein the term fats is also used as a generic term for such liquid fats. Fats include: plant oils (for example: Apricot Kernel Oil, Arachis Oil, Arnica Oil, Argan Oil, Avocado Oil, Babassu Oil, Baobab Oil, Black Seed Oil, Blackberry Seed Oil, Blackcurrant Seed Oil, Blueberry Seed Oil, Borage Oil, Calendula Oil, Camelina Oil, Camellia Seed Oil, Castor Oil, Cherry Kernel Oil, Cocoa Butter, Coconut Oil, Corn Oil, Cottonseed Oil, Evening Primrose Oil, Grapefruit Oil, Grapeseed Oil, Hazelnut Oil, Hempseed Oil, Jojoba Oil, Lemon Seed Oil, Lime Seed Oil, Linseed Oil, Kukui Nut Oil, Macadamia Oil, Maize Oil, Mango Butter, Meadowfoam Oil, Melon Seed Oil, Moringa Oil, Olive Oil, Orange Seed Oi l, Pa l m Oi l, Pa paya Seed Oi l, Passion Seed Oi l, Peach Kernel Oi l, Pl um Oil, Pomegranate Seed Oil, Poppy Seed Oil, Pumpkins Seed Oil, Rapeseed (or Canola) Oil, Red Raspberry Seed Oil, Rice Bran Oil, Rosehip Oil, Safflower Oil, Seabuckthorn Oil, Sesame Oil, Soyabean Oil, Strawberry Seed Oil, Sunflower Oil, Sweet Almond Oil, Walnut Oil, Wheat Germ Oil); fish oils (for example: Sardine Oil, Mackerel Oil, Herring Oil, Cod-liver Oil, Oyster Oil); animal oils (for example: Conjugated Linoleic Acid); or other oils (for example: Paraffinic Oils, Naphthenic Oils, Aromatic Oils, Silicone Oils); or any mixture thereof.

Iodine Value As used herein the term "iodine value" is used as a generic term for the measure of the unsaturation of oil and is expressed in terms of the number of centigrammes of iodine absorbed per gramme of sample (% iodine absorbed). The higher the iodine number, the more unsaturated double bonds are present in oil and hence the more prone the oil is to oxidisation via the double bond. Iodine value is determined using the Wijs Method as provided in the American Oil Chemists' Society (AOCS) Official Method Tg 1 a-64, pages 1-2, Official Methods and Recommended Practices of the American Oil Chemists' Society, Second Edition, edited by D. Firestone, AOCS Press, Champaign, 1990, method Revised 1990).

Calculation of ratio of hydrophobin to oil

As used herein the term "ratio of hydrophobin to oil" is defined as the mass of hydrophobin (in grammes) relative to the volume of the oil (in litres) in the oil-in-water emulsion. The ratio of hydrophobin to oil is therefore expressed as:

Total mass of Hydrophobin in emulsion (grammes) : Total volume of oil in emulsion (litres) = g/litre

Calculation of ratio of oil to water

As used herein the term "ratio of oil to water" is defined as the volume of oil (in mi Mi l itres) relative to the volume of the water (i n m i 11 i I it res) i n the oi l-in-water emulsion. The ratio of oil to water is therefore expressed as:

(Total volume of oil in emulsion (millilitres) / Total volume of water in emulsion (millilitres) ) x 100 = v/v%

Oil-in-water emulsion

As used herein the term "oil-in-water emulsion" is used as a generic term for a mixture of two immiscible phases wherein an oil (dispersed phase) is dispersed in an aqueous solution (the continuous phase). Food Products

As used herein the term "food products" is used as a generic term for products and ingredients taken by the mouth, the constituents of which are active in and/or absorbed by the gastrointestinal tract with the purpose of nourishment of the body and its tissues, refreshment and indulgence, which products are to be marketed and sold to customers for consumption by humans. Examples of food products are tea, including precursors thereof; spreads; ice cream; frozen fruits and vegetables; snacks including diet foods and beverages; condiments; dressings; and culinary aids. Food products may particularly bring any of the following benefits: healthy metabolism; life span extension; optimal growth and development; optimal gastrointestinal tract function; avoidance of metabolic syndrome and insulin resistance; avoidance of dyslipidemias; weight control; healthy mineral metabolism; immune health; optimal eye health; avoidance of cognitive impairment and memory loss; hair and skin health; beauty; and excellent taste and smell.

Spreads

As used herein the term "spreads" is used as a generic term for oi l a nd water containing emulsion, for instance a margarine type spread. Advantageously a spread has a pH of 4.8-6.0. The pH can be measured by metlting the spread, separating the molten fat phase from the water phase and measuring the pH of the water phase.

Spreads of the invention may comprise other ingredients commonly used for spreads, such as flavouring ingredients, thickeners, gellation agents, colouring agents, vitamins, emulsifiers, pH regulators, stabilizers etc. Common amounts of such ingredients as well as suitable ways to prepare margarines or spreads are well-known to the skilled person.

Dressings

As used herein the term "dressings" is used as a generic term for oil and water containing emulsion, for instance vinaigrette and salad-dressing type compositions. Aeration

The term "aerated" means that gas has been intentionally incorporated into the product, such as by mechanical means. The gas can be any gas, but is preferably, particularly in the context of food products, a food-grade gas such as air, nitrogen or carbon dioxide. The extent of aeration is typically defined in terms of "overrun". In the context of the present invention, %overrun is defined in volume terms as:

Overrun =

((volume of the final aerated product - volume of the mix) / volume of the mix)

X 100 The amount of overrun present in the product will vary depending on the desired product characteristics. For example, the level of overrun in confectionery such as mousses can be as high as 200 to 250%. The level of overrun in some chilled products, ambient products and hot products can be lower, but generally over 10%, e.g. the level of overrun in milkshakes is typically from 10 to 40%.

Hydrophobins

Hydrophobins are a well-defined class of proteins (Wessels, 1997, Adv. Microb. Physio. 38: 1-45; Wosten, 2001, Annu Rev. Microbiol. 55: 625-646) capable of self-assembly at a hydrophobic/hydrophilic interface, and having a conserved sequence: Xn-C-X5-9-C-C-X11-39-C-X8-23-C-X5-9-C-C-X6-18-C-Xm (SEQ ID No. 1) where X represents any amino acid, and n and m independently represent an integer. Typically, a hydrophobin has a length of up to 125 amino acids. The cysteine residues (C) in the conserved sequence are part of disulphide bridges. In the context of the present invention, the term hydrophobin has a wider meaning to include functionally equivalent proteins still displaying the characteristic of self-assembly at a hydrophobic- hydrophilic interface resulting in a protein film, such as proteins comprising the sequence:

Xn-C-XI-SO-C-XO-S-C-XI-IOO-C-XI-IOO-C-XI-SO-C-XO-S-C-XI-S O-C-Xm (SEQ ID No. 2) or parts thereof still displaying the characteristic of self-assembly at a hydrophobic- hydrophilic interface resulting in a protein film. In accordance with the definition of the present invention, self-assembly can be detected by adsorbing the protein to Teflon and using Circular Dichroism to establish the presence of a secondary structure (in general, α-helix) (De Vocht et al., 1998, Biophys. J. 74: 2059-68).

The formation of a film can be established by incubating a Teflon sheet in the protein solution followed by at least three washes with water or buffer (Wosten et al., 1994,

Embo. J. 13: 5848-54). The protein film can be visualised by any suitable method, such as labeling with a fluorescent marker or by the use of fluorescent antibodies, as is well established in the art. m and n typically have values ranging from 0 to 2000, but more usually m and n in total are less than 100 or 200. The definition of hydrophobin in the context of the present invention includes fusion proteins of a hydrophobin and another polypeptide as well as conjugates of hydrophobin and other molecules such as polysaccharides.

Hydrophobins identified to date are generally classed as either class I or class II. Both types have been identified in fungi as secreted proteins that self-assemble at hydrophobilic interfaces into amphipathic films. Assemblages of class I hydrophobins are relatively insoluble whereas those of class Il hydrophobins readily dissolve in a variety of solvents.

Hydrophobin-like proteins have also been identified in filamentous bacteria, such as Actinomycete and Steptomyces sp. (WO01/74864). These bacterial proteins, by contrast to fungal hydrophobins, form only up to one disulphide bridge since they have only two cysteine residues. Such proteins are an example of functional equivalents to hydrophobins having the consensus sequences shown in SEQ ID Nos. 1 and 2, and are within the scope of the present invention.

The hydrophobins can be obtained by extraction from native sources, such as filamentous fungi, by any suitable process. For example, hydrophobins can be obtained by culturing filamentous fungi that secrete the hydrophobin into the growth medium or by extraction from fungal mycelia with 60% ethanol. It is particularly preferred to isolate hydrophobins from host organisms that naturally secrete hydrophobins. Preferred hosts are hyphomycetes (e.g. Trichoderma), basidiomycetes and ascomycetes. Particularly preferred hosts are food grade organisms, such as Cryphonectria parasitica which secretes a hydrophobin termed cryparin (MacCabe and Van Alfen, 1999, App. Environ. Microbiol 65: 5431-5435).

Alternatively, hydrophobins can be obtained by the use of recombinant technology. For example host cells, typically micro-organisms, may be modified to express hydrophobins and the hydrophobins can then be isolated and used in accordance with the present invention. Techniques for introducing nucleic acid constructs encoding hydrophobins into host cells are well known in the art. More than 34 genes coding for hydrophobins have been cloned, from over 16 fungal species (see for example WO96/41882 which gives the sequence of hydrophobins identified in Agaricus bisporus; and Wosten, 2001 , Annu Rev. Microbiol. 55: 625-646). Recombinant technology can also be used to modify hydrophobin sequences or synthesise novel hydrophobins having desired/improved properties.

Typically, an appropriate host cell or organism is transformed by a nucleic acid construct that encodes the desired hydrophobin. The nucleotide sequence coding for the polypeptide can be inserted into a suitable expression vector encoding the necessary elements for transcription and translation and in such a manner that they will be expressed under appropriate conditions (e.g. in proper orientation and correct reading frame and with appropriate targeting and expression sequences). The methods required to construct these expression vectors are well known to those skilled in the art.

A number of expression systems may be used to express the polypeptide coding sequence. These include, but are not limited to, bacteria, fungi (including yeast), insect cell systems, plant cell culture systems and plants all transformed with the appropriate expression vectors. Preferred hosts are those that are considered food grade - 'generally regarded as safe' (GRAS).

Suitable fungal species, include yeasts such as (but not limited to) those of the genera Saccharomyces, Kluyveromyces, Pichia, Hansenula, Candida, Schizo saccharomyces and the like, and filamentous species such as (but not limited to) those of the genera Aspergillus, Trichoderma, Mucor, Neurospora, Fusarium and the like. The sequences encoding the hydrophobins are preferably at least 80% identical at the amino acid level to a hydrophobin identified in nature, more preferably at least 95% or 100% identica l . H owever, persons ski l led i n the a rt may ma ke conservative substitutions or other amino acid changes that do not reduce the biological activity of the hydrophobin. For the purpose of the invention these hydrophobins possessing this high level of identity to a hydrophobin that naturally occurs are also embraced within the term "hydrophobins".

Hydrophobins can be purified from culture media or cellular extracts by, for example, the procedure described in WO01/57076 which involves adsorbing the hydrophobin present in a hydrophobin-containing solution to surface and then contacting the surface with a surfactant, such as Tween 20, to elute the hydrophobin from the surface. See also Collen et al., 2002, Biochim Biophys Acta. 1569: 139-50; Calonje et al., 2002, Can. J. Microbiol. 48: 1030-4; Askolin et al., 2001, Appl Microbiol Biotechnol. 57: 124-30; and De Vries et al., 1999, Eur J Biochem. 262: 377-85.

Shelf-life

As used herein the term "shelf-life" is used as a generic term for the length of time that a consumer product such as a food product may be considered suitable for sale or consumption. In particular, shelf-life is the time that products can be stored, during which the defined quality of a specified proportion of the goods remains acceptable under expected (or specified) conditions of distribution, storage and display. In the instant case, shelf-l ife refers to the length of time that a n oil-in-water emulsion maintains the physio-chemical properties critical for ensuring consumer acceptance of these products.

Brief Description of the Invention

We have now found that oil-in-water emulsions that are resistant to oxidisation may be obtained in formulations comprising certain amounts of hydrophobin and oil.

Accordingly, in a first aspect, the present invention provides an oil-in-water emulsion comprising hydrophobin and oil wherein the oil-in-water emulsion has an overrun of less than 35% and wherein the oil has an iodine value of greater than 40 characterised in that the ratio of hydrophobin to oil is greater than 20g/litre, the ratio of oil to water being greater than 1 v/v% preferably greater than 2 v/v%, more preferably greater than 4 v/v%, more preferably still greater than 5 v/v%, yet more preferably greater than 7 v/v%, most preferably greater than 15 v/v%.

Having conducted extensive research into the stabilisation of oil-in-water emulsions and the prevention of degradation thereof we have found that the advantage of the ratio of hydrophobin to oil is that in such emulsions oxidisation of oil in the dispersed phase is significantly reduced. Accordingly the ratio of hydrophobin to oil is preferably greater than 30g/litre, more preferably greater than 40g/litre, more preferably still greater than 60g/litre and most preferably greater than 80g/litre.

As set out a bove, the hyd rophobi n may be a class I or a class Il hydrophobin, preferably a class Il hydrophobin, more preferably the hydrophobin is HFBII.

The i nvention is pa rticula rly su itable to unaerated oi l-in-water emulsions and accordingly, the overrun is preferably less than 25%, more preferably less than 20%, more preferably still less than 10%, most preferably less than 5%.

The invention is capable of preventi ng the degradation of oxidisa ble oils a nd accordingly in a preferred embodiment the iodine value of the oil is greater than 60, more preferably greater than 90, more preferably still greater than 120, most preferably greater than 140.

Particular oils are especially suitable for use according to the invention and accordingly the oil is preferably selected from the group consisting of olive oil, corn oil, canola oil, soybean oil, sunflower oil, linseed oil, and any mixture thereof.

In a second aspect, the present invention provides food products comprising an oil-in- water emulsion comprising hydrophobin and oil wherein the oil-in-water emulsion has an overrun of less than 35% and wherein the oil phase has an iodine value of greater than 40 characterised in that the ratio of hydrophobin to oil is greater than 20g/litre, the ratio of oil to water being greater that 1 v/v%, preferably greater than 2 v/v%, more preferably greater than 4 v/v%, more preferably still greater than 5 v/v%, yet more preferably greater than 7 v/v%, most preferably greater than 15 v/v%.

The products are preferably food products wherein the food products are selected from the group consisting of dressings, ice creams, mayonnaises, spreads and sauces.

Detailed description of the invention

Examples Hydrophobin

HFBII (Mw = 7200 g.mol-1) solution from VTT Biotechnology Finland was used for all experiments. Unless otherwise stated, all water used for the experiments was of Millipore quality.

ONs

Purified sunflower oil (SFO) was used for all experiments. The composition and Iodine Value of the sunflower oil used is given in Table 1.

Table 1 - Com osition and Iodine Value of Sunflower Oil

Preparation of Hydrophobin (HFB) and Whey Protein Isolate (WPI) solutions pH adjusted Double Distilled Water (DDW) at pH 2, pH 3 and pH 7 was prepared using 0.1 M HCI or 0.1 M NaOH. HFB or WPI was then incorporated into each pH adjusted DDW to a concentration of 0.2 wt% and the pH of the final solutions with each protein were adjusted with 0.1 M HCI or NaOH to pH 2, 3 or 7. To these solutions, SFO was added and homogenized as follows.

Preparation of emulsions Compositions according to Table 2 were first subjected to Ultraturrax at 6, 500 RPM for 1 min, followed by 24,000 RPM for 10 min with continuous shaking of the beaker and then carefully transferred to Microfluidizer (MF) in order not to introduce unnecessary foam formation a nd subjected to M F at 1000 bar for 5 mi n with ice to prevent temperature increase. Bottles of emulsions were then sealed and pasteurized at 80 ⁰ C for 10 minutes and then allowed to cool. The emulsions were unaerated and thus had an overrun of 25%.

Table 2 - Composition of emulsions

Accelerated oxidation test

An accelerated oxidation test was performed on the pH 2, 3 and 7 emulsions of Comparative Example 1 and Example A as provided below. The accelerated oxidation test was carried out over 42 days at 40 ⁰ C because the period and temperature are representative of a period of from 9 to 12 months at a temperature of 20 ⁰ C. The accelerated oxidation test assesses the p rog ress of oxidation th roug h the measurement of volatile components resulting from oxidation and was performed as follows:

1. 1 ml aliquots of Comparative Example 1 and Example A were separated into individual vials.

2. The air in the headspace of the vials was flushed out with nitrogen gas.

3. The vials were sealed with a cap with a rubber septum.

4. All vials were placed in an incubator at 40 ⁰ C in the absence of light for 42 days. 5. After the 42 day experimental period the vials were removed from the incubator and cooled in the absence of light to allow hexanal to dissolve back into the emulsion. After cooling, the head space of the vials was again flushed with nitrogen gas. 6. Gas Chromatography (GC) was performed to detect volatile components. During GC, the vials were heated to 60 ⁰ C to release volatile components from the emulsion into the headspace which was subsequently measured using GC. The levels of volatile components detected using GS was expressed as peak area as calculated from the chromatograms. Among the various volatiles, hexanal is the most common representative of oxidation and the results therefore present only Hexanal.

The results of the oxidation test for 42 days at 40 ⁰ C as shown in Tables 3 - 5 show that HFB stabilized emulsions are less oxidized than WPI stabilized emulsions.

Table 3 - Results of accelerated oxidation test of pH 2 emulsions after 42 days

Table 4 - Results of accelerated oxidation test of pH 3 emulsions after 42 days

Table 5 - Results of accelerated oxidation test of pH 7 emulsions after 42 days